Phosphorylation of smooth muscle myosin II (SMM) and nonmuscle myosin II (NMM) is required to activate cellular contractile functions. Phosphorylation of a single serine in the regulatory light chain (RLC) of each of the two "head" domains of myosin is sufficient to activate the ATPase by more than 1000 fold (1). The current knowledge suggests that this remarkable regulatory switch is mediated through large conformational changes in the myosin structure. Our long-term goal is to determine the structural basis of the phosphorylation-dependent regulatory switch. We have demonstrated that both head domains (2) and both catalytic domains (3) of SMM and NMM are required to retain an intact regulatory switch. However, atomic resolution structures of double-headed myosin constructs have not been forthcoming, even after more than 10 years since the initial myosin head structure was solved (4). And, all atomic resolution structures of single-head constructs have limited or no structural information about the RLC and no information about the RLC domain in which the phosphorylated serine is found. There are no structures available for the RLC of SMM or NMM. To address this problem, we have applied transient kinetic (1,5,6), fluorescence (7), electron paramagnetic resonance (EPR) (8) and photo-crosslinking (5,9,10) approaches toward an understanding of the structural basis of the regulatory switch. We have shown that 1) there is an interaction between the two RLC in the switched-off state that appears to be weakened or altered in the phosphorylated switched-on state (9,10), 2) the N-terminal region of the regulatory domain (RD) containing serine-19 appears to undergo a large conformational change from an extended to folded structure (10) and 3) this conformational change requires the presence of nucleotide at the active site approximately 15 nm away (7). Hypotheses We have published an atomic resolution structural model of the RD of SMM in the switched-off state (10), which serves as one of our initial structural hypotheses for this proposal. Here we extend this initial work to propose the Hydrogen Bonding Network Hypothesis to explain how the regulatory domain switches between the on- and off-states. We also propose a structural hypothesis to explain how RLC phosphorylation destabilizes the full-length SMM 10S conformation. Aims We have devised a series of experiments that directly test the above-mentioned structural hypotheses in addition to structural proposals by others (11-13). We propose to use a truncated myosin construct (HMM) and full-length myosin, both of which are capable of adopting a fully switched-off and fully switched-on conformation. We also address key unanswered questions about the effect of phosphorylation upon the kinetic mechanism of ATP hydrolysis.